Purification procedures such a precipitation, molecular sieve chromatography, electrophoresis, affinity chromatography and covalent chromatography are well known in the art and have been utilized in the purification of proteins from cell extracts. However, purification of proteins produced by cells transformed by recombinant DNA sequences that code for them has posed unique and difficult problems.
Preferably, the level of expression of the recombinant DNA sequence is high and therefore the host cell transformed by that DNA sequence produces a large amount of the desired protein within the cell. Accordingly, the tranformed cell accumulates large numbers of foreign protein molecules. These molecules may then interact with each other to form highly insoluble aggregates, not typically found in the normal cell. The host cell then responds to this unusual accumulation of foreign protein by forming inclusion bodies composed of the foreign protein aggregates. Purification of these foreign proteins in a biologically active form, therefore, requires a means of solubilizing these highly insoluble protein aggregates in such a way to preserve or to enable recovery of their native conformation and a means for purifying the soluble protein in a manner that maintains the biological activity of the protein.
A genetically engineered protein of great value to the health field is interferon. ln this application we will use the interferon nomenclature announced in Nature, 286, p. 110 (Jul. 10, 1980). "IFN" will designate interferon, "IFN-.alpha." will designate leukocyte interferon, "IFN-.beta." will designate fibroblast interferon, and "IFN-.gamma." will designate gamma interferon.
IFN is a cellular protein displaying antiviral activity against a broad range of viruses through induction of cellular RNA and protein synthesis directed against virus replication. For example, human IFN has been used to combat the viral activity of the following: respiratory infections; [Texas Reports on Biology and Medicine, Vol. 35, pp. 486-96 (1977) (hereinafter referred to as Texas Reports)]; herpes simplex keratitis [Texas Reports, pp. 497-500; R. Sundmacher, "Exogenous Interferon In Eye Diseases", International Virology IV, The Hague, Abstract nr. w2/11, p. 99 (1978)]; acute hemorrhagic conjunctivitis [Texas Reports, pp. 501-510]; adenovirus keraton conjunctivitis [A. Romano et al., ISM MemoI-A8131 (October, 1979)]; varicella-zoster [Texas Reports, pp. 511-515]; cytomegalovirus infection [Texas Reports, pp. 523-527]; and hepatitis B [Texas Reports, pp. 516-522]. See also W. E. Stewart, II, The Interferon System, pp. 307-321 Springer-Verlag (2 ed.) (1981) (hereinafter referred to as The Interferon System).
IFN has other effects in addition to its antiviral action. For example, it antagonizes the effect of colony stimulating factor, inhibits the growth of hemopoietic colony-forming cells and interferes with the normal differentiation of granulocyte and macrophage precursors [Texas Reports, pp. 343-349]. It also inhibits erythroid differentiation in DMSO-treated Friend leukemia cells [Texas Reports, pp. 420-428].
IFN may also play a role in the regulation of the immune response. For example, depending upon the dose and time of application in relation to antigen, IFN can be both immunopotentiating and immunosuppressive in vivo and in vitro [Texas Reports, pp. 357-369]. In addition, IFN is also known to enhance the activity of killer lymphocytes and antibody-dependent cell-mediated cytotoxicity [R. R. Herberman et al., "Augmentation By Interferon of Human Natural And Antibody-Depenent Cell-Mediated Cytotoxicity", Nature, 227, pp. 221-223 (1979); P. Beverley and D. Knight, "Killing Comes Naturally", Nature, 278, pp. 119-120 (1979); Texas Reports, pp. 375-380; J. R. Huddlestone et al., "Induction and Kinetics of Natural Killer Cells In Humans Following Interferon Therapy", Nature, 282, pp. 417-419 (1979); S. Einhorn et al., "Interferon And Spontaneous Cytotoxicity In Man. II. Studies In Patients Receiving Exogenous Leukocyte Interferon", Acta Med. Scand., 204, pp. 478-83 (1978).
Killer lymphocytes and antibody-dependent cell-mediated cytotoxicity may be directly or indirectly involved in the immunological attack on tumor cells. Therefore, in addition to its use as an antiviral agent, IFN has potential application in antitumor and anticancer therapy and in immunomodulation agents and methods [The Interferon System, pp. 319-21, 250-56. It is now known that IFNs affect the growth of many classes of tumors in many animals [The Interferon System, pp. 292-304.]Interferons, like other antitumor agents, seem most effective when directed against small tumors. The antitumor effects of animal IFN are dependent on dosage and time, but have been demonstrated at concentrations below toxic levels. Accordingly, numerous investigations and clinical trials have been and continue to be conducted into the antitumor and anticancer properties of human IFNs. These include treatment of several malignant diseases such as osteosarcoma, acute myeloid leukemia, multiple myeloma and Hodgkin's disease [Texas Reports, pp. 429-35.] Although the reuslts of these clinical tests are encouraging, the antitumor, anticancer and immunomodulation applications of human IFNs have been severely hampered by lack of an adequate supply of purified IFN.
Interferons have been classified into two groups: Type I and Type II IFNs. Type I IFNs are the "classical" acid stable IFNs induced by viruses or synthetic polynucleotides and generally consist of two species: IFN-.alpha. and IFN-.beta.. Type II IFN consists of only one species designated as IFN-.gamma., also referred to in the art as gamma interferon.
IFN-.gamma. is a glycoprotein induced in lymphocytes by specific antigen or various mitogens and is antigenically distinct from IFN-.alpha. and IFN-.beta.. [A. Mizrahi et al., "Glycosylation Of Interferon", J. Biol. Chem., 253, pp. 7612-15 (1978); The Interferon System, pp. 107-08; P. Gray et al., "Expression Of Human Immune Interferon cDNA In E. coli And Monkey Cells", Nature, 295, 503-08 (1982); M. P. Langford et al., "Large-Scale Production And Physicochemical Characterization Of Human Immune Interferon", Infection And Immunity, 26, pp. 36-41 (1979)]. The protein has been reported to have a molecular weight of 40,000-46,000 daltons, with the possibility that its glycosylated form has a molecular weight of 65,000-70,000 daltons. In addition to being acid labile (at pH2), IFN-.gamma. has been reported to be inactivated after 1 hour at 56.degree. C. See also M. DeLey et al., "Interferon Induced In Human Leukocytes By Mitogens: Prodcution, Partial Purification and Characterization," Eur. J. Immunol., 10, pp. 877-83 (1980); Y. K. Yip et al., "Partial Purification and Characterization Of Human .gamma. (Immune) Interferon", Proc. Natl. Acad. Sci. USA, 78, pp. 1601-05 (1981). It has been reported that IFN-.gamma. recognizes a different cell receptor than IFN-.alpha. or IFN-.beta. [A. A. Branca et al. "Evidence That Types I And II Interferons Have Different receptors", Nature, 294, pp. 768-70 (1981)].
In addition to its antiviral activity, IFN-.gamma. is reported to display antitumor activity. Moreover, as compared to IFN-.alpha. and IFN-.beta., IFN-.gamma.'s antitumor activity seems, at least in mice, to result in tumor regression. In addition, its activation of natural killer cells does not reach a plateau, as observed for IFN-.alpha. and IFN-.beta., and IFN-.gamma. appears to be less inhibited by circulating levels of gangliosides than are IFN-.alpha. and IFN-.beta.[H. Ankel et al., "Mouse Fibroblast (Type I) And Immune (Type II) Interferons: Pronounced Differences In Affinity for Gangliosides and In antiviral And Antigrowth Effects On Mouse Leukemia L-1210R Cells", Proc. Natl. Acad. Sci. USA. 77, pp. 2528-32 (1980). Therefore, it appears that cells or tumors that display poor response to IFN-.alpha. or IFN-.beta. may be effectively treated with IFN-.gamma. [e.g., Crane et al., J. Natl. Cancer Institute, 61, p. 891 (1978); Barn et al., Abstract N.Y. Acad. Sci., No. II (Oct. 23-26, 1979; Blalock et. al., Cellular Immunology, 49, pp, 390-94 (1980); B. Y. Rubin et al., "Differential Efficacies Of Human Type I And Type II Interferons As Antiviral And Antiproliferative Agents", Proc. Natl. Acad. Sci. USA, 77, pp. 5928-32 (1980).
Furthermore, it has been suggested that the primary function of IFN-.gamma. may be as an immunoregulatory agent. The antiproliferative effect of IFN-.gamma. on transformed cells has been reported to be 10 to 100 times greater than that of IFN-.alpha. or IFN-.beta. [P. W. Gray et al., supra].
Several techniques for the purification of human IFN-.gamma. from human cells have been disclosed. One such technique involves the purification of IFN-.gamma. from cultures of human leukocytes by sequential adsorptions on controlled-pore glass (CPG) and concanavalin A-Sepharose followed by an adsorption on DEAE-Sephacel [Y. K. Yip et al., "Purification of Two Species Of Human .gamma. (lmmune) lnterferon", Proc. Natl. Acad. Sci. USA, 79, pp. 1820-24 (1982)]. Another technique is described in U.S. Pat. No. 4,382,027 issued to I. A. Braude on May 3, 1983. IFN-.gamma. is purified to near homogeneity from mitogen-induced human peripheral blood leukocytes by sequential adsorptions on CPG beads, concanavalin A-Sepharose, lentil lectin-Sepharose or pea lectin-agarose, and Heparin-Sepharose or Procian Red-agarose followed by gel filtration chromatography. See also M. P. Langford, supra; M. Wiranowska-Stewart et al., "Production, Partial Purification and Characterization of Human and Murine Interferons - Type II, Molecular Immunology, 12, pp. 623-25 (1980); M. deLey et al., supra; Y. K. Yip et al., "Partial Purification And Characterizatiuon of Human .gamma. (Immune) Interferon,"Proc. Natl. Acad. Sci USA, 78, pp. 1601-05 (1981); J. A. Georgiades, "Production and Purification of the Human Interferon Gamma (HuIFN-.gamma.)", Texas Reports on Biology and Medicine, Vol. 41, pp. 179-83 (1981-82).
Standard methods for the purification of human IFN-.gamma. from human cells require the induction or stimulation of the cells by antigens or mitogens in order to produce sufficient amounts of IFN-.gamma. for purification. See J. A. Georgiades, supra. Even with these purification procedures, however, it is not possible to produce sufficient amounts of IFN-.gamma. for large scale use in clinical trials or in antiviral, antitumor, anticancer or immunomodulation methods and agents.
The technique of genetic engineering, whereby the DNA sequences coding for human IFN-.gamma. are cloned and expressed in a host cell, allows the production of large amounts of the protein. However, as it true for many genetically engineered proteins, the human IFN-.gamma. produced in the host is in the form of highly insoluble protein aggregates. It has proven, therefore, very difficult to isolate and to purify the IFN-.gamma. from the extracts of the variety of hosts in which it it produced. These purification problems have prevented IFN-.gamma. from becoming available in the amounts needed for use in antiviral, anticancer and immunomodulation methods and agents.